This invention relates to the fields of hematology and immunology and to reagents used for analyzing blood cells. More specifically, the present invention relates to an improved diluent which produces less than about 1 part per million of formaldehyde during both usage and storage.
Common medical diagnostic procedures involve the analysis of a blood sample of a patient in order to make certain clinical diagnoses. For example, complete blood counts, analyses of the white and red cell numbers and/or subpopulations, and the presence of unusual cell types as detected by a variety of flow cytometric assays provide valuable information for the physician. Regardless of the subsequent steps to be performed in any of the well-known diagnostic analytic procedures for blood samples, the first step is generally a dilution of the blood sample. Specifically, the blood sample is diluted with a diluent that may contain a mixture of salts, buffers, and preservatives, among others. See, e.g., the discussion of the contents and problems of prior art diluents in U.S. Pat. No. 5,935,857, for example.
However, the use of many commercially available blood diluents has been restricted due to the regulation of institutional and industrial, including medical, wastes. For example, regulatory agencies in the states of California and Massachusetts, for example, are increasingly requiring reduced concentrations of formaldehyde in wastewater. Unfortunately, at present, most of the commercially available blood diluents produce formaldehyde in amounts in excess of about 400 parts per million, or if there is an absence of formaldehyde producing components, the diluent contains other undesirable components, such as sodium azide, or does not have the broad biocidal protection.
There exists a need in the art for a multipurpose blood diluent which produces considerably less than about 400 parts per million of formaldehyde and does not negatively impact the analysis of samples, has broad biocidal activity and does not contain undesirable components.
The present invention is directed toward blood diluents which produce less than about 1 part per million of formaldehyde. The present invention is also directed toward such substantially formaldehyde-free diluents, which maintain good antimicrobial activity, but do not adversely affect essential parameters of blood analysis, such as mean corpuscular volume (MCV) and other blood cell parameters.
The present invention is further directed toward methods of analyzing a blood sample containing blood cells using the blood diluents of the invention.
These and other aspects of the invention will be understood to one of skill in the art upon reading of the following detailed description of the invention.
The present invention resolves the problem of formaldehyde production by blood diluents by providing a novel blood diluent which contains less than about 1 part per million of formaldehyde. Further, methods for analyzing the blood samples to determine at least one physical parameter of the blood sample using the blood diluents of the invention are provided.
I. The Diluent Composition
The present invention provides a low formaldehyde-containing aqueous blood diluent containing a first component selected from specified potentiator reagents and a second component which is a combination of an effective anti-microbial amount of 5-chloro-2-methyl-4-isothiazolin-3-one, 2-methyl-4-isothiazolin-3-one; and 5-bromo-5-nitro-1,3-dioxane. The compositions are combined in concentrations and with other components that permit less than about 1 parts per million of formaldehyde to be produced in the diluent.
The phrase xe2x80x9clow formaldehydexe2x80x9d as used herein and throughout this specification is meant to describe a solution having or producing less than about 1 part per million (ppm) of formaldehyde over a period of time of at least 1 year. In one embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.70 ppm of formaldehyde. In another embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.60 ppm of formaldehyde. In a further embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.50 ppm of formaldehyde. In yet another embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.40 ppm of formaldehyde. In another embodiment, xe2x80x9clow of formaldehydexe2x80x9d describes a solution having or producing less than about 0.30 ppm of formaldehyde. In yet a further embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.20 ppm of formaldehyde. In another embodiment, xe2x80x9clow formaldehydexe2x80x9d describes a solution having or producing less than about 0.10 ppm of formaldehyde.
The inventors have recently discovered that preservative agents contribute significantly to the production of formaldehyde in commercially available blood diluents. Specifically, the widely used preservative agent dimethylolurea (DMOU) has been found to produce greater than 1 part per million of formaldehyde both alone and in combination with other reagents. Since the blood diluents of the invention produce less than about 1 ppm formaldehyde, it is preferable for reagents that produce or contain greater than 1 ppm of formaldehyde to be omitted from the composition of the diluent of the invention. Since the inventors have discovered that dimethylolurea produces greater than 1 ppm formaldehyde, it is preferably completely excluded from the diluent composition of the invention.
As used herein and throughout this specification, an xe2x80x9ceffective amountxe2x80x9d of a component is selected with regard to the pH, osmolality, concentration, conductivity and/or antimicrobial efficacy of the final diluent composition and with respect to the function of the particular component. One of skill in the art would readily be able to determine the effective amount of each component. While specific ranges are noted, alternate amounts may also be contemplated by one of skill in the art.
The term xe2x80x9caqueousxe2x80x9d as used herein and throughout this specification is meant to describe a solution having water as its main component. The solution may additionally contain other chemical compounds and/or solvents that do not interfere with the effectiveness of the diluent. Such additional solvents may be selected by one of skill in the art and include, but are not limited to, organic solvents, inorganic solvents, and saline solutions.
A. Potentiator Reagents
As described herein, the present invention is directed toward a low formaldehyde-containing aqueous blood diluent that contains one or more potentiators as a first component. Without wishing to be bound by theory, the blood diluent potentiator is a compound that chelates ions in the cell membrane and weakens the membrane, making the cell more susceptible to biocides. In one embodiment, one potentiator may be ethylenediamine tetraacetic acid (EDTA), an EDTA derivative, or combinations thereof. In another embodiment, the potentiator is an EDTA derivative. For example, in an embodiment, the potentiator is disodium EDTA. In another embodiment, the potentiator is dipotassium EDTA. In still another embodiment, the potentiator is ethyleneglycol-bis-(2-aminoethyl ether)N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA). More preferably, the potentiator is disodium EDTA.
In another embodiment, additional potentiators other than EDTA or its derivatives may be added to the diluent. Such additional potentiators include potentiators that chelate ions as described above, which are known to those skilled in the art. Some of these potentiators include, without limitation, cyclohexanediamine-tetraacetic acid, diethylentriaminepentaacetic acid, and/or hydroxyethylethylene-diaminetriacetic acid. Still other potentiators which function via mechanisms other than chelation are known and may also be mixed with a chelating potentiator into the biocidal formulation of the diluent.
The concentration of each potentiator in the diluent is typically present from about 0.05 grams per liter (g/L) to about 2.0 g/L. Preferably, the concentration of the potentiator is from about 0.05 to about 1.5 g/L.
The term xe2x80x9cderivativexe2x80x9d as used herein and throughout this specification is meant to describe a compound that is structurally similar to the parent compound. Derivatives may include, but are not limited to, salts that may be derived from pharmaceutically or physiologically acceptable inorganic or organic acids and bases, and organically acceptable variations thereof.
B. Antimicrobial Agents
Diagnostic reagents may be stored for extended periods while during use and manufacture, and can often be exposed to microorganisms. Thus, the diluent of this invention must exhibit antimicrobial robustness, since the presence of such microorganisms contaminating the diluent can distort the analysis of the sample. Antimicrobial agents must be utilized to eliminate growth of microorganisms prior to and during analysis. The term xe2x80x9cantimicrobial agentxe2x80x9d as used herein and throughout this specification is meant to describe any chemical compound that is effective in reducing or eliminating microorganisms including, but not limited to, gram positive bacteria, gram negative bacteria, fungi, and yeast. However, as noted above, antimicrobial agents used in the diluents known in the art have been found by the inventors to at least contribute to the production of greater than 1 ppm of formaldehyde in the diluent. For use in the diluent of this invention, antimicrobial agents must have the following characteristics: In association with other components of the diluent composition, these reagents must produce little or insubstantial amounts (e.g., preferably, less than 1 ppm) of formaldehyde. The reagents must also provide adequate antimicrobial protection and yet not interfere with the measurement of the blood sample in the various analytical procedures. Preferably, the antimicrobial agents do not react with lytic, or other, reagents conventionally used in many methods of blood analysis.
The blood diluent of the invention thus contains one or more antimicrobial agents which are effective in eliminating gram negative bacteria, gram positive bacteria, yeast, and fungi, and yet which produce in the diluent composition less than 1 ppm formaldehyde. Preferably, the antimicrobial agents useful in the diluent of the invention include various combinations of 5-chloro-2-methyl-4-isothiazolin-2-one, 2-methyl-4-isothiazolin-3-one, and 5-bromo-5-nitro-1,3-dioxane and derivatives of each of these compounds.
These antimicrobial agents are commercially available. The 5-chloro-2-methyl-4-methyl-4-isothiazolin-2-one and 2-methyl-4-isothiazolin-3-one are supplied in combination commercially by Supelco, under the trademark Proclin. For example, aqueous combinations of these antimicrobial agents include the Proclin(copyright) 150 reagent (Supelco), which is an aqueous mixture of 1.15% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.35% of 2-methyl-4-isothiazolin-3-one, and the Proclin(copyright) 300 reagent (Supelco), which is a mixture of 2.3% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of 2-methyl-4-isothiazolin-3-one in a solvent consisting of a modified glycol and alkyl carboxylate. Another product supplied commercially by the same company is called Proclin(copyright) 5000 reagent, which contains only 2-methyl-4-isothiazolin-3-one in a dipropylene glycol solvent. The biocide 5-bromo-5-nitro-1,3-dioxane is commercially available and include the Bronidox(copyright) L reagent (Cognis Corporation).
When added as neat reagents, neat 5-chloro-2-methyl-4-isothiazolin-2-one and neat 2-methyl-4-isothiazolin-3-one are present in the diluent at a combined concentration of about 0.0015 to about 0.030 g/L at a ratio of about 1: about 0.3 (5-chloro-2-methyl-4-isothiazolin-2-one: 2-methyl-4-isothiazolin-3-one). As used herein and throughout this specification, the term xe2x80x9cneatxe2x80x9d is meant to describe a reagent alone, without additional solvents or reagents. The fixed ratio of the two components as provided in the commercial Proclin(copyright) reagents have been shown to be acceptable for use in the diluent of this invention. However, other ratios of these two components may also be determined by one of skill in the art. Additionally, useful individual concentrations of the two components may be calculated readily from these preparations. More preferably, neat 5-chloro-2-methyl-4-isothiazolin-2-one and neat 2-methyl-4-isothiazolin-3-one may be added to the diluent of the invention and are present in the diluent at a combined concentration of about 0.005 to about 0.01 g/L. In another embodiment, 5-chloro-2-methyl-4-isothiazolin-2-one and neat 2-methyl-4-isothiazolin-3-one may be added to the diluent of the invention as mixtures that are commercially available. When antimicrobial agents are added as a mixture, the Proclin(copyright) 150 reagent may preferably be present in the diluent at a concentration of about 0.1 to about 2.0 xcexcL. More preferably, the Proclin(copyright) 150 reagent may be present in the diluent at a concentration of about 0.25 to about 1.0 xcexcL. The present invention is however not bound by the specific source of these antimicrobial agents or concentrations of the Proclin(copyright) reagents.
The other antimicrobial agent, 5-bromo-5-nitro-1,3-dioxane functions in the diluent to assist in eliminating gram-positive bacteria and provide other broad biocidal activity against other organisms and does not produce or contain greater than 1 ppm of formaldehyde when employed in the blood diluent of the invention. In one embodiment, neat 5-bromo-5-nitro-1,3-dioxane is employed in the diluent at a concentration of about 0.010 to about 0.50 g/L and preferably from about 0.015 to about 0.30 g/L. In another embodiment, the Bronidox(copyright) L reagent is employed as the source of 5-bromo-5-nitro-1,3-dioxane and is present in the diluent at a concentration of about 0.1 to about 2.0 g/L. More preferably, the Bronidox(copyright) L reagent may be present in the diluent at a concentration of about 0.5 to about 1.5 g/L.
When employed in combination in the diluent formulation of this invention, these three anti-microbial compounds may be employed in any ratio effective to combat microbial growth, without producing formaldehyde, and further without adversely effecting the blood sample or blood cell parameter to be measured by the selected analytical procedures. Preferably, the ratio is that of the fixed ratio of the Proclin(copyright) 150 product with the above-indicated amounts of the Bronidox(copyright) L reagent. Useful individual ratios of the three components may be calculated readily from these preparations. However, other ratios of these three components may also be determined by one of skill in the art.
Given the requirements of for multipurpose blood diluents set out by the present inventors in the instant specification, it is anticipated that other antimicrobials may be similarly useful in diluent compositions of this invention. Selection of such useful antimicrobial components following the teachings contained herein is encompassed by this invention.
C. Other Diluent Components
The diluent of the present invention may also contain other components that produce less than 1 ppm formaldehyde in combination with the first three components. Preferably these other components produce little or no formaldehyde in combination with reagents commonly used in blood sample analysis, e.g., lytic reagents, and do not interfere with the analysis of the blood samples.
In one embodiment, the diluent may contain as an added component, an alkali metal sulfate. Without wishing to be bound by theory, the alkali metal sulfate regulates the osmolality and ionic strength of the diluent. Preferably, the alkali metal sulfate includes, but is not limited to, sodium and potassium sulfates. More preferably, the alkali metal sulfate is sodium sulfate. In one embodiment, the alkali metal sulfate is added to the diluent as an aqueous solution. In another embodiment, the alkali metal sulfate is added to the diluent as a solid. Preferably, the alkali metal sulfate is present in the diluent at a concentration of about 7 to about 14 g/L. More preferably, the alkali metal sulfate is present in the diluent at a concentration of about 8 to about 12 g/L.
The diluent may also contain as an additional component, an alkali metal chloride. Without wishing to be bound by theory, the alkali metal chloride assists the alkali metal sulfate in regulating the osmolality and ionic strength of the diluent. Preferably, the alkali metal chloride includes, but is not limited to, sodium and potassium chlorides. More preferably, the alkali metal is sodium chloride. In one embodiment, the alkali metal chloride is added to the diluent as an aqueous solution. In another embodiment, the alkali metal chloride may be added to the diluent as a solid. Preferably, the alkali metal chloride is present at a concentration of about 2 to about 6 g/L. More preferably, the alkali metal chloride is present at a concentration of about 3 to about 5 g/L.
The diluent may additionally contain a base. Without wishing to be bound by theory, the base is added to the diluent in an amount sufficient to adjust the pH of the diluent to the preferred range. Adjustment of the amount of the base to provide a suitable pH is based upon the buffer employed and is within the skill of the art. Preferably, the base is an alkali metal hydroxide, however other bases may be utilized provided that they do not interfere with the analysis of the blood sample and may be selected by one of skill in the art. More preferably, the alkali metal hydroxide includes sodium and potassium hydroxide. Most preferably, the alkali metal hydroxide is sodium hydroxide. In one embodiment, the base is added as an aqueous solution. In another embodiment, the base is added as an aqueous 50% sodium hydroxide solution. In a further embodiment, the base is added to the diluent as a solid.
The desired pH of the diluent depends upon the hematology instrument utilized and the test to be performed. Preferably, the diluent will be adjusted such that the pH of the diluent is from about 5 to about 8. More preferably, the diluent will be adjusted such that the pH of the diluent is from about 6 to about 7.5. Even more preferably, the diluent will be adjusted such that the pH of the diluent is from about 6.5 to about 7.6. Sodium hydroxide base is present in the diluent at a concentration sufficient enough to achieve the desired pH. This concentration is dependent on the concentration of other solutes that may contribute to the overall acidity or basicity of the diluent solution. One of skill in the art may readily determine the amount of base required.
The diluent may also contain as a desirable component, a cell stabilizing agent that prevents platelet aggregation. An exemplary cell stabilizing agent is an anesthetic. Preferably, the anesthetic is ethyl p-aminobenzoate hydrochloride. However, other anesthetics or cell stabilizing agents may be selected and utilized in the diluent by one of skill in the art following the teachings of this invention. In one embodiment, neat ethyl p-aminobenzoate hydrochloride is added to the diluent. In another embodiment, the Procaine(copyright) hydrochloride reagent (Aceto Corp.) may be added as the source of ethyl p-aminobenzoate hydrochloride. Preferably, ethyl p-aminobenzoate hydrochloride is present in the diluent at a concentration of about 0.05 to about 0.25 g/L. More preferably, ethyl p-aminobenzoate hydrochloride is present in the diluent at a concentration of about 0.07 to about 0.15 g/L.
Additionally, the diluent may contain a buffer. Without wishing to be bound by theory, the buffer primarily maintains the pH of the diluent. In one embodiment, the buffer is N-(2-acetoamido)iminodiacetic acid (ADA). This buffer has also been reported to assist in lysing erythrocyte debris, and stabilizing the size distribution and cellular shape of erythrocytes and platelets. However, other buffers such as the Good buffers identified in Good, N. E. et al. (1966) Biochemistry 5, 467 and Good, N. E., and Izawa, S. (1972) Methods Enzymol. 24, 53 may be utilized depending upon the functional requirements of the formulation as determined by one skilled in the art. Preferably, N-(2-acetoamido)iminodiacetic acid is present in the diluent at a concentration of about 1.0 to about 2.5 g/L. More preferably, N-(2-acetoamido)iminodiacetic acid is present in the diluent at a concentration of about 1.2 to about 2.0 g/L.
The diluent may further contain other reagents including preservative agents, anticoagulants, detergents, dyes, and stains. See, for example, U.S. Pat. Nos. 5,935,857 and 4,528,274. However, any additional compounds added to the diluent must not increase the formaldehyde production nor adversely impact the physical parameters that are analyzed.
In one preferred embodiment, a diluent of this invention contains disodium ethylenediamine tetraacetic acid, 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one, 5-bromo-5-nitro-1,3-dioxane, sodium chloride, sodium sulfate, sodium hydroxide, ethyl p-aminobenzoate hydrochloride, and N-(2-acetoamido)iminodiacetic acid in suitable amounts to produce less than about 1 part per million of formaldehyde, to perform suitably as an antimicrobial, and to perform suitably as a blood diluent or sheath fluid.
Still another particularly desirable diluent composition according to this invention is illustrated in Table 1, below:
The diluent composition as described in Table 1 produces less than about 1 part per million of formaldehyde, to perform suitably as an antimicrobial, and to perform suitably as a blood diluent or sheath fluid.
These and other diluents of the present invention do not degrade during the shelf life of the product. The shelf life of the product takes into account the time from manufacture of the product through the time of customer usage of the product. Preferably, the diluent has a shelf life greater than 1 month. More preferably, the diluent has a shelf life greater than 3 months. Most preferably, the diluent has a shelf life greater than 12 months. Based on additional recent data, it is anticipated that the diluent has a shelf life of 18 months or more.
The diluents according to this invention may also be used over a wide range of operating temperatures. The diluent may be frozen and, once thawed and thoroughly mixed, performs satisfactorily. The diluent has been heated to about 150xc2x0 F. for up to 36 days with no deterioration of performance. The diluent is typically used over a temperature range from about 60xc2x0 F. to about 90xc2x0 F. consistent with the allowable operating temperature range of Coulter hematology instruments.
Generally, the diluent will be iso-osmotic. Preferably, the osmolality is from about 250 to about 350 milliOsmo/kg (mOsm/kg). More preferably, the osmolality will be from about 300 to about 340 mOsm/kg. However, the osmolality of the diluent can vary when used it is with a lytic reagent composition. The volume of the diluent can however be adjusted relative to a lytic reagent volume to effect an optimum final osmolality of the blood sample mixture.
In addition, when used as a sheath fluid in flow cytometry analysis, the relationship between the osmolality and conductivity of the sheath fluid and the osmolality and conductivity of the core fluid should be maintained. For example, the diluent will typically have a conductivity from about 16 to 22 mS/cm.
Still another advantage of the low formaldehyde diluents of this invention is that analytic parameters of the blood sample diluted with a diluent of this invention are comparable to measurements obtained when using a commercially available diluent. For example, the diluents of the present invention provide stable MCV for fresh and aged blood as demonstrated in the examples below. Other parameters of blood analysis should remain undistorted by use of this diluent composition.
II. Methods of Using the Blood Diluent Composition
The multipurpose blood diluent of the present invention is useful in a variety of known analytic methods performed on a diluted blood sample to determine at least one physical parameter of the blood sample. In addition, the diluent of the present invention is useful as a sheath fluid for the analysis of blood samples in a focus flow instrument. More particularly, the diluent of the present invention has background fluorescence suitable for use in a fluorescence measurement of a body fluid. Suitable blood samples may be obtained from a veterinary or human patient and include, but are not limited to, whole blood, plasma, serum and urine.
Blood samples may be fresh, i.e. tested within about 8 hours from phlebotomy, or the samples may be aged for longer periods of time up to 72 hours either at room temperature or refrigerated. Preferably, aged blood samples may be analyzed within 48 hours either at room temperature or refrigerated. Most preferably, aged blood samples may be analyzed within 24 hours either at room temperature or refrigerated.
For example, the diluent is useful in methods for the determination of hematologic parameters of the blood samples. Such parameters include one or more of cell size, shape, content and volume and may include white blood cell count (WBC), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), Plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), neutrophils (NE), lymphocytes (LY) monocytes (MO), eosinophils (EO), basophils (BA), and reticulocytes (RET).
Such measurements may be made using light scatter, low frequency current, radio frequency current, fluorescence and combinations thereof. The diluent is also conveniently used as a sheath fluid in focused flow cytometry for differentiating white blood cells into three or more subpopulations, or for determination of five subpopulations of leukocytes, and for fluorescence flow cytometry analysis when using fluorescence probes or antibodies. For example, U.S. Pat. Nos. 5,223,398 and 5,231,005 describe various methods for producing a five-part differential of white blood cells into lymphocyte, monocyte, neutrophil, eosinophil, and basophil classes. This five-part differential is accomplished in automated equipment that detects differences in cell volume (V), cell conductivity (C), and light scatter (S). One of skill in the art can readily select other methods for analysis of blood samples that can make use of the diluent compositions of this invention. It is understood that the advantages of the diluent compositions of this invention make it a desirable component of many such analytic procedures, both automated or non-automated.
The following examples are provided to illustrate the production and activity of representative compounds of the invention and to illustrate their performance in a screening assay. One skilled in the art will appreciate that although specific reagents and conditions are outlined in the following examples, these reagents and conditions are not a limitation on the present invention.